羊草Class Ⅱ几丁质酶基因的克隆及序列分析

Molecular Cloning and Sequence Analysis of Class Ⅱ Chitinase Gene in Leymus chinensis

[目的]克隆自然生长于黑龙江省盐碱地的羊草ClassⅡ几丁质酶基因并进行序列分析,为进一步研究几丁质酶基因的生物功能和应用奠定了基础。[方法]构建羊草叶片的cDNA文库,对其进行DNA序列测定和分析,并与GenBank中收录的植物几丁质酶基因序列及编码的氨基酸序列进行同源性比较。[结果]在羊草叶片cDNA文库中克隆出1条全长cDNA片段,片段长996 bp,其中,可读框768 bp,编码255个氨基酸。编码产物在结构上缺乏CBD和C-端延伸区,具有ClassⅡ几丁质酶的结构特征,其氨基酸序列与黑麦和小麦ClassⅡ几丁质酶具有很高的同源性;构建的pQE-LcChi2重组载体经诱导后表达出一个约27 KD的蛋白,与推测的pQE-LcChi2基因编码产物大小一致。[结论]LcChi2基因在大肠杆菌中能够表达,是一个具有表达功能的基因。

[ Objective] The aim was to clone Class II chitinase gene in Leymus chinensis which are growing in saline land in Heilongjiang Province, and lay a foundation for further researching the biological function and application of chitinase gene. [ Method ] Constructing cDNA library of Leymus chinensis leaves,its DNA sequence was determined and analyzed, the homology of chitinase gene and amino acid sequence was compared with that in the GeneBank. [ Result] The total length cDNA segment was cloned,the length was 996 bp, the ORF was 768 bp among which, encoding 255 amino acids. It had the structural character of Class Ⅱ chitinase gene because the encoded products lacked CBD and C- terminal extension region. The amino acid sequence had high homology compared with Class I chitinase genc of rye and wheat. The constructed recombinant vector pQE-LcChi2 can induce and express a protein of 27 KD, the result was consistent with deduced encoded product of pQE-Lc- Chi2 gene. [ Conclusion ] LcChi2 gene has expressed function, which can express in E. coll.